Vascular endothelial growth factor (VEGF) molecule and process for producing same

ABSTRACT

The present invention relates generally to an isolated molecule having vascular endothelial growth factor-like properties and to a genetic sequence encoding same. The molecule will be useful in the development of a range of therapeutics and diagnostics useful in the treatment, prophylaxis and/or diagnosis of conditions requiring enhanced or diminished vasculature and/or vascular permeability. The molecule of the present invention is also a useful effector of primary and central neurons and is capable of inducing astroglial proliferation.

[0001] The present invention relates generally to an isolated moleculehaving vascular endothelial growth factor-like properties and to agenetic sequence encoding same. The molecule will be useful in thedevelopment of a range of therapeutics and diagnostics useful in thetreatment, prophylaxis and/or diagnosis of conditions requiring enhancedor diminished vasculature and/or vascular permeability. The molecule ofthe present invention is also a useful effector of primary and centralneurons and is capable of inducing astroglial proliferation.

[0002] Bibliographic details of the publications referred to by authorin this specification are collected at the end of the description.Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and aminoacid sequences referred to in the specification are defined followingthe bibliography.

[0003] Throughout this specification, unless the context requiresotherwise, the word “comprise”, or variations such as “comprises” or“comprising”, will be understood to imply the inclusion of a statedelement or integer or group of elements or integers but not theexclusion of any other element or integer or group of elements orintegers.

[0004] Vascular endothelial growth factor (hereinafter referred to as“VEGF”), also known as vasoactive permeability factor, is a secreted,covalently linked homodimeric glycoprotein that specifically activatesendothelial tissues (Senger et al., 1993). A range of functions havebeen attributed to VEGF such as its involvement in normal angiogensisincluding formation of the corpus luteum (Yan et al., 1993) andplacental development (Sharkey et al., 1993), regulation of vascularpermeability (Senger et al., 1993), inflammatory angiogenesis(Sunderkotter et al., 1994) and autotransplantation (Dissen et al.,1994) and human diseases such as tumour promoting angiogenesis (Folkman& Shing, 1992), rheumatoid arthritis (Koch et al., 1994) and diabetesrelated retinopathy (Folkman & Shing, 1992). VEGF is, therefore, animportant molecule making it a potentially valuable target for researchinto therapeutics, prophylactics and diagnostic agents based on VEGF orits activities. There is also a need to identify homologues or otherwiserelated molecules for use as an alternative to VEGF or in conjunctionwith VEGF.

[0005] In work leading up to the present invention, the inventors soughtthe multiple endocrine neoplasia type I susceptibility gene (MEN1).Surprisingly, the inventors discovered that a genetic sequence excludedas a candidate for the MEN1 gene was nevertheless a new growth factorhaving some similarity to VEGF. Furthermore, the growth factor of thepresent invention is an effector molecule for primary and centralneurons.

[0006] Accordingly, one aspect of the present invention comprises abiologically isolated proteinaceous molecule comprising a sequence ofamino acids which:

[0007] (i) is at least about 15% similar to the amino acid sequence setforth in SEQ ID NO:2; and

[0008] (ii) is at least 5% dissimilar to the amino acid sequence setforth in SEQ ID NO:2.

[0009] Another aspect of the present invention provides a biologicallyisolated proteinaceous molecule having the following characteristics:

[0010] (i) comprises an amino acid sequence having at least about 15%similarity but at least about 5% dissimilarity to all or part of theamino acid sequence set forth in SEQ ID NO:2;

[0011] (ii) exhibits at least one property in common with VEGF.

[0012] A related aspect of the present invention contemplates abiologically isolated proteinaceous molecule having the followingcharacteristics:

[0013] (i) comprises an amino acid sequence having at least about 15%similarity but at least about 5% dissimilarity to the amino acidsequence set forth in SEQ ID NO:2;

[0014] (ii) exhibits at least one of the following properties:

[0015] (a) ability to induce proliferation of vascular endothelialcells;

[0016] (b) ability to interact with flt-1/flk-1 family of receptors;

[0017] (c) ability to induce cell migration, cell survival and/or anincrease in intracellular levels of alkaline phosphatase.

[0018] By “biologically isolated” is meant that the molecule hasundergone at least one step of purification from a biological source.Preferably, the molecule is also biologically pure meaning that acomposition comprises at least about 20%, more preferably at least about40%, still more preferably at least about 65%, even still morepreferably at least about 80-90% or greater of the molecule asdetermined by weight, activity or other convenient means, relative toother compounds in the composition. Most preferably, the molecule issequencably pure.

[0019] Another preferred aspect of the present invention provides themolecule in recombinant form.

[0020] According to this aspect of the present invention, there isprovided a recombinant molecule comprising a sequence of amino acidswhich:

[0021] (i) is at least about 15% similar to the amino acid sequence setforth in SEQ ID NO:2; and

[0022] (ii) is at least 5% dissimilar to the amino acid sequence setforth in SEQ ID NO:2.

[0023] A related aspect of the present invention is directed to arecombinant molecule having the following characteristics:

[0024] (i) comprises an amino acid sequence having at least about 15%similarity but at least about 5% dissimilarity to all or part of theamino acid sequence set forth in SEQ ID NO:2;

[0025] (ii) exhibits at least one property in common with VEGF.

[0026] A further related aspect of the present invention contemplates arecombinant molecule having the following characteristics:

[0027] (i) comprises an amino acid sequence having at least about 15%similarity but at least about 5% dissimilarity to the amino acidsequence set forth in SEQ ID NO:2;

[0028] (ii) exhibits at least one of the following properties:

[0029] (a) ability to induce proliferation of vascular endothelialcells;

[0030] (b) ability to interact with flt-1/flk-1 family of receptors;

[0031] (c) ability to induce cell migration, cell survival and/or anincrease in intracellular levels of alkaline phosphatase.

[0032] The present invention also contemplates genomic or partial genomeclones encoding a proteinaceous molecule having at least about 15% aminoacid similarity but at least about 5% dissimilarity to SEQ ID NO:1.

[0033] The amino acid sequence set forth in SEQ ID NO:2 corresponds tohuman VEGF (referred to herein as “VEGF₁₆₅”). Accordingly, the moleculeof the present invention is VEGF-like or is a homologue of VEGF butcomprises an amino acid sequence which is similar but non-identical tothe amino sequence of VEGF. Although the present invention isexemplified using a human VEGF-like molecule, this is done with theunderstanding that the instant invention contemplates the homologousmolecule and encoding sequence from other mammals such as livestockanimals (e.g. sheep, pigs, horses and cows), companion animals (e.g.dogs and cats) and laboratory test animals (e.g. mice, rats, rabbits andguinea pigs) as well as non-mammals such as birds (e.g. poultry birds),fish and reptiles. In a most preferred embodiment, the VEGF-likemolecule is of human origin and encoded by a gene located at chromosome11q13. The present invention extends, therefore, to the genomic sequenceor part thereof encoding the subject VEGF-like molecule.

[0034] Preferably, the percentage similarity is at least about 30%, morepreferably at least about 40%, still more preferably at least about 50%,still even more preferably at least about 60-70%, yet even morepreferably at least about 80-95% to all or part of the amino acidsequence set forth in SEQ ID NO:2.

[0035] In a particularly preferred embodiment, the VEGF-like molecule ofthe present invention comprises a sequence of amino acids as set forthin SEQ ID NO:4 or is a part, fragment, derivative or analogue thereof.Particularly preferred similarities include about 19-20%, and 29-30%.Reference herein to derivatives also includes splice variants.Accordingly, the present invention extends to splice variants of SOM175.Examples of splice variants contemplated by the present inventioninclude but are not limited to variants with an amino acid sequencesubstantially as set forth in at least one of SEQ ID NO:6, SEQ ID NO:8and/or SEQ ID NO:10 or mutants or derivatives or further splice variantsthereof.

[0036] Another embodiment provides a recombinant molecule having thefollowing characteristics:

[0037] (i) an amino acid sequence substantially as set forth in SEQ IDNO:4 or having at least about 15% similarity to all or part thereofprovided that said amino acid sequence is at least about 5% dissimilarto all or part of the amino acid sequence set forth in SEQ ID NO:2;

[0038] (ii) exhibits at least one biological property in common withVEGF.

[0039] Another embodiment provides a recombinant molecule having thefollowing characteristics:

[0040] (i) an amino acid sequence substantially as set forth in SEQ IDNO:6 or having at least about 15% similarity to all or part thereofprovided that said amino acid sequence is at least about 5% dissimilarto all or part of the amino acid sequence set forth in SEQ ID NO:2;

[0041] (ii) exhibits at least one biological property in common withVEGF.

[0042] Another embodiment provides a recombinant molecule having thefollowing characteristics:

[0043] (i) an amino acid sequence substantially as set forth in SEQ IDNO:8 or having at least about 15% similarity to all or part thereofprovided that said amino acid sequence is at least about 5% dissimilarto all or part of the amino acid sequence set forth in SEQ ID NO:2;

[0044] (ii) exhibits at least one biological property in common withVEGF.

[0045] Another embodiment provides a recombinant molecule having thefollowing characteristics:

[0046] (i) an amino acid sequence substantially as set forth in SEQ IDNO:10 or having at least about 15% similarity to all or part thereofprovided that said amino acid sequence is at least about 5% dissimilarto all or part of the amino acid sequence set forth in SEQ ID NO:2;

[0047] (ii) exhibits at least one biological property in common withVEGF.

[0048] Such properties of VEGF include at least one of:

[0049] (a) ability to induce proliferation of vascular endothelialcells;

[0050] (b) an ability to interact with flt-1/flk-1 family of receptors;

[0051] (c) an ability to induce cell migration, cell survival and/or anincrease in intracellular levels of alkaline phosphatase.

[0052] In accordance with the present invention, a preferred similarityis at least about 40%, more preferably at least about 50% and even morepreferably at least about 65% similarity.

[0053] Still a further aspect of the present invention contemplates apeptide fragment corresponding to a portion of the amino acid sequenceset forth in SEQ ID NO:4 or a splice variant thereof such as set forthin SEQ ID NO:6, SEQ ID NO:8 or SEQ ID NO:10 or a chemical equivalentthereof. The biologically isolated or recombinant molecule of thepresent invention may be naturally glycosylated or may comprise analtered glycosylation pattern depending on the cells from which it isisolated or synthesised. For example, if produced by recombinant meansin prokaryotic organisms, the molecule would be non-glycosylated. Themolecule may be a full length, naturally occurring form or may be atruncated or otherwise derivatised form.

[0054] Yet another aspect of the present invention is directed to anucleic acid molecule encoding the VEGF-like molecule herein described.More particularly, the present invention provides a nucleic acidmolecule comprising a sequence of nucleotides substantially as set forthin SEQ ID NO:3 or having at least 15% similarity to all or part thereofor being capable of hybridising under low stringency conditions to areverse complement of the nucleotide sequence as set forth in SEQ IDNO:3 provided that the nucleic acid sequence having at least 15%similarity but at least 30% dissimilarity to the nucleotide sequence asset forth in SEQ ID NO:3. The nucleotide sequence set forth in SEQ IDNO:3 is also referred to herein as “SOM175”. Preferably, the percentagedissimilarity is about 35%, more preferably about 39% and even morepreferably about 40-50% or greater.

[0055] For the purposes of defining the level of stringency, referencecan conveniently be made to Sambrook et al (1989) at pages 9.47-9.51which is herein incorporated by reference where the washing stepsdisclosed are considered high stringency. A low stringency is definedherein as being in 4-6×SSC/0.1-0.5% w/v SDS at 37-45° C. for 2-3 hours.Depending on the source and concentration of nucleic acid involved inthe hybridisation, alternative conditions of stringency may be employedsuch as medium stringent conditions which are considered herein to be1-4×SSC/0.25-0.5% w/v SDS at ≧45° C. for 2-3 hours or high stringentconditions considered herein to be 0.1-1×SSC/0.1% w/v SDS at 60° C. for1-3 hours.

[0056] The present invention further contemplates a nucleic acidmolecule which encodes a VEGF-like molecule as hereinbefore describedhaving at least 15% nucleotide sequence homology to SEQ ID NO:3.Preferred levels of homology include at least about 40%, more preferablyaround 60-70%.

[0057] The present invention is further directed to the murine homologueof human VEGF (referred to herein as “mVRF”). The mVRF has approximately85% identity and 92% conservation of amino acid residues over the entirecoding region compared to human VEGF. The mVRF is encoded by a nucleicacid molecule comprising a nucleotide sequence substantially as setforth in FIG. 9.

[0058] The VEGF-like molecule of the present invention will be useful inthe development of a range of therapeutic and/or diagnostic applicationsalone or in combination with other molecules such as VEGF. The presentinvention extends, therefore, to pharmaceutical compositions comprisingthe VEGF-like molecule or parts, fragments, derivatives, homologues oranalogues thereof together with one or more pharmaceutically acceptablecarriers and/or diluents. Furthermore, the present invention extends tovectors comprising the nucleic acid sequence set forth in SEQ ID NO:3 orhaving at least about 15%, more preferably about 40% and even morepreferably around 60-70% similarity thereto but at least 30% and morepreferably around 39% dissimilarity thereto and host cells comprisingsame. In addition, the present invention extends to ribozymes andantisense molecules based on SEQ ID NO:3 as well as neutralizingantibodies to the VEGF-like molecule. Such molecules may be useful inameliorating the effects of, for example, over expression of VEGF-likegenes leading to angiogenesis or vascularization of tumours.

[0059] Another aspect of the present invention contemplates a method ofinducing astroglial proliferation in a mammal, said method comprisingadministering to said mammal an effective amount of a recombinantproteinaceous molecule having the characteristics:

[0060] (i) comprises an amino acid sequence having at least about 15%similarity but at least about 5% dissimilarity to the sequence set forthin SEQ ID NO:2;

[0061] (ii) exhibits at least one property in common with vascularendothelial growth factor (VEGF),

[0062] said administration being for a time and under conditionssufficient to induce astroglial proliferation.

[0063] Preferably, the recombinant proteinaceous molecule comprises theamino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:6.

[0064] A further aspect of the present invention provides a method ofpromoting neural survival and/or proliferation in a mammal, said methodcomprising administering to said mammal an effective amount of arecombinant proteinaceous molecule having the characteristics:

[0065] (i) comprises an amino acid sequence having at least about 15%similarity but at least about 5% dissimilarity to the sequence set forthin SEQ ID NO:2;

[0066] (ii) exhibits at least one property in common with vascularendothelial growth factor (VEGF),

[0067] said administration being for a time and under conditionssufficient to induce astroglial proliferation.

[0068] Preferably, the recombinant proteinaceous molecule comprises theamino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:6.

[0069] The present invention also contemplates antibodies to theVEGF-like molecule or nucleic acid probes to a gene encoding theVEGF-like molecule which are useful as diagnostic agents.

[0070] The present invention is further described by reference to thefollowing non-limiting Figures and/or Examples.

[0071] In the Figures:

[0072]FIG. 1 Nucleotide sequence [SEQ ID NO:1] and corresponding aminoacid sequence [SEQ ID NO:2] of VEGF₁₆₅.

[0073]FIG. 2 Nucleotide sequence [SEQ ID NO:3] and corresponding aminoacid sequence [SEQ ID NO:4] of SOM175.

[0074]FIG. 3 Results of BLAST search with SOM175 protein sequence.

[0075]FIG. 4 BESTFIT alignment of VEGF cDNA and SOM175 cDNA.

[0076]FIG. 5 Multiple alignment of VEGF₁₆₅ with SOM175 and its splicevariants at the nucleotide level.

[0077]FIG. 6 Multiple alignment of VEGF₁₆₅ with SOM175 and its splicevariants at the amino acid level.

[0078]FIG. 7 Diagrammatic representation of SOM175 and its splicevariants.

[0079]FIG. 8(a) Diagrammatic representation of genomic structure ofhuman SOM175 genomic showing exon/intron map.

[0080]FIG. 8(b) Diagrammatic representation of genomic structure ofhuman SOM175 showing exon/intron boundries.

[0081]FIG. 9 Nucleotide and predicted peptide sequences derived frommVRF cDNA clones. Numbering of nucleotides are given on the left,starting from the A of the initiation codon. Amino acids are numbered onthe right, starting from the first residue of the predicted matureprotein after the putative signal peptide has been removed. Thealternately spliced region is double underlined and the resultingpeptide sequence from each mRNA is included. A potential polyadenylationsignal is indicated in boldface. Start and stop codons of mVRF₁₆₇ andmVRF₁₈₆ are underlined and a polymorphic AC repeat in the 3′ UTR isindicated by a stippled box. The positions of intron/exons boundariesare indicated by arrowheads.

[0082]FIG. 10 BESTFIT alignments of human and murine VRF proteinisoforms. A: mVRF₁₆₇ and hVRF₁₆₇. B: mVRF₁₈₆ and hVRF₁₈₆ from the pointwhere the sequences diverge from the respective 167 amino acid isoforms.Amino acid identities are marked with vertical bars and conserved aminoacids with colons. An arrow marks the predicted signal peptide cleavagesite of human and mouse VRF.

[0083]FIG. 11 BESTFIT alignment of mVRF₁₆₇ and mVEGF₁₈₈ (Breier et al,1992) peptide sequences. An arrow marks the signal peptide cleavage siteof mVEGF. Identical amino acids are indicated by vertical bars andconservative substitutions by colons. Numbering of amino acids is asdescribed in the legend to FIG. 9.

[0084]FIG. 12 Comparison of gene structure between VRF (a generic VRFgene is shown since the intron/exon organisation of the mouse and humanhomologues is almost identical) and other members of the humanVEGF/PIGF/PDGF gene family. Exons are represented by boxes. Proteincoding regions and untranslated regions are shown by filled and opensections respectively. The hatched region in VRF indicates theadditional 3′ UTR sequence formed by alternate splicing of the VRF₁₈₆isoform. Potential alternate splice products of each gene are shown.

[0085]FIG. 13 Autoradiogram of a Northern blot of total RNA from variousadult mouse tissues (as indicated) hybridised with an mVRF cDNA clone. Amajor transcript of 1.3 kb was detected in all samples.

[0086]FIG. 14 Film autoradiographs (A-C) and dark-field micrographs(D-E) illustrating the expression pattern of mVRF and mRNA in the mouse.In the E14 mouse embryo (A) positive signals are present over thedeveloping heart (Ha) and cerebral cortex (Cx). A low background signalis also present over other tissues in the section. In the E17 embryo (B)and the heart (Ha) is clearly visible due to a strong hybridisationsignal. An equally strong signal is present over brown adipose tissue(Fa) in the back and around the thoracic cage. A moderate hybridisationsignal is present over the spinal cord (SC) and the tongue (T). Thebackground signal is reduced compared with the E14 embryo. In the youngadult mouse (C-D), positive signals are present over the heart (Ha) andadipose tissue (Fa) around the thoracic cage, while, for example, thelungs (Lu) are unlabeled). The hybridisation signal over the heart isevenly distributed over the entire left ventricle, including papillarymuscles (D). In the E17 heart hybridised with an excess of cold probe,no positive signal is present (E). Scale bars=0.5 mm (A), 1.2 mm (B), 1mm (C), 0.3 mm (D), 0.1 mm (E).

[0087]FIG. 15 Dark—(A and C) and bright-field (B and D) micrographsshowing mVRF mRNA expression in mouse adipose tissue (A-B) and spinalcord (C-D). A strong hybridisation signal is present over fat (A), asshown by the strong labeling in Sudan black stained sections (B). A weaksignal is present also in skeletal muscle (M in A-B). In the adultspinal cord (C) the mVRF probes gave a neuronal staining pattern overthe gray matter. Toloudine counterstaining showing that motoneurons inthe ventral horn (D), interneurons in the deep part of the dorsal hornand around the central canal (not shown) where largely positive for mVRFmRNA. Scale bars=0.1 mm (A), 0.1 mm (B), 0.25 mm (C), 0.015 mm (D).

[0088]FIG. 16 Effect of VEGF on embryonic day 8 (E8) chick sensoryneurons as determined by % survival, % neurite outgrowth and averageneurite length (μm).

[0089]FIG. 17 Effects of VEGF and SOM175 on chick glia. Tested were CNSglial, peripheral glia and CNS oligodendrocytes.

[0090]FIG. 18 Effect of various SOM175 proteins on mouse astroglialcells. ▪ ³H (cpm)

[0091] 1. FGF-2 (10 ng/ml) positive control

[0092] 2. SOMΔX6* 1 ng/ml

[0093] 3. SOMΔX6 10 ng/ml

[0094] 4. SOMΔX6 100 ng/ml

[0095] 5. SOMΔX6 1000 ng/ml

[0096] 6. SOMAΔX6 1000 ng/ml, no heparin

[0097] 7. SOMX6** 1 ng/ml

[0098] 8. SOMX6 10 ng/ml

[0099] 9. SOMX6 100 ng/ml

[0100] 10. SOMX6 1000 ng/mI

[0101] 11. SOMX6 1000 ng/ml, no heparin

[0102] * This refers to SOM175 absent exon 6;

[0103] ** This refers to SOM175.

[0104]FIG. 19 Effect of various SOM175 proteins on mouse oligodenroglialcells. ▪ ³H (cpm)

[0105] 1. FGF-2 (10 ng/ml) positive control

[0106] 2. SOMΔX6* 1 ng/ml

[0107] 3. SOMΔX6 10 ng/ml

[0108] 4. SOMΔX6 100 ng/ml

[0109] 5. SOMΔX6 1000 ng/ml

[0110] 6. SOMΔX6 1000 ng/ml, no heparin

[0111] 7. SOMX6** 1 ng/ml

[0112] 8. SOMX6 10 ng/ml

[0113] 9. SOMX6 100 ng/ml

[0114] 10. SOMX6 1000 ng/ml

[0115] 11. SOMX6 1000 ng/ml, no heparin

[0116] * This refers to SOM175 absent exon 6;

[0117] ** This refers to SOM175.

[0118]FIG. 20 Effect of various SOM175 proteins on mouse forebrainneurons. ▪% survival

[0119] 1. FGF-2 (10 ng/ml) positive control

[0120] 2. SOMΔX6* 1 ng/ml

[0121] 3. SOMΔX6 10 ng/ml

[0122] 4. SOMΔX6 100 ng/ml

[0123] 5. SOMΔX6 1000 ng/ml

[0124] 6. SOMΔX6 1000 ng/ml, no heparin

[0125] 7. SOMX6** 1 ng/ml

[0126] 8. SOMX6 10 ng/ml

[0127] 9. SOMX6 100 ng/ml

[0128] 10. SOMX6 1000 ng/ml

[0129] 11. SOMX6 1000 ng/ml, no heparin

[0130] * This refers to SOM175 absent exon 6;

[0131] ** This refers to SOM175. TABLE 1 SUMMARY OF SEQUENCE IDENTITYNUMBERS SEQ ID NO: 1 Nucleotide sequence of VEGF₁₆₅ SEQ ID NO: 2 Aminoacid sequence of VEGF₁₆₅ SEQ ID NO: 3 Nucleotide sequence of SOM175(VEGF-like molecules) SEQ ID NO: 4 Amino acid sequence of SOM175 SEQ IDNO: 5 Nucleotide sequence of SOM175 absent exon 6 SEQ ID NO: 6 Aminoacid sequence of SOM175 absent exon 6 SEQ ID NO: 7 Nucleotide sequenceof SOM175 absent exon 6 and exon 7 SEQ ID NO: 8 Amino acid sequence ofSOM175 absent exon 6 and exon 7 SEQ ID NO: 9 Nucleotide sequence ofSOM175 absent exon 4 SEQ ID NO: 10 Amino acid sequence of SOM175 absentexon 4 SEQ ID NO: 11 Oligonucleotide SEQ ID NO: 12 Oligonucleotide SEQID NO: 13 Oligonucleotide SEQ ID NO: 14 Oligonucleotide

EXAMPLE 1

[0132] Human cDNA Clones

[0133] The original SOM175 cDNA was isolated by screening a human foetalbrain library (λzapII, Stratagene) with the cosmid D11S750 (Larsson etal, 1992). The plasmid was excised “in vivo” and a single 1.1 kb cDNAwas obtained. Three independent SOM175 cDNAs clones were also isolatedfrom a human foetal spleen library (Strategane, Unizap) using theabove-mentioned SOM175 insert as a probe. Three clones were obtained:SOM175-4A, -5A and -6A. SOM175-5A is an alternately spliced clone withexon 4 being absent (SOM175-e4). These library screens were performedusing hybridisation conditions recommended by the manufacturer of thelibrary (Stratagene) and random primed insert of SOM175.

[0134] Two partial human SOM175 cDNAs have also isolated from a λGT11human melanoma cell line A2058 library (Clontech) cDNA library screenswere performed using hybridisation conditions described by Church andGilbert, 1984). In each case, the probe was generated by random primingof a PCR product derived from SOM175 (18f-700r).

[0135] Mouse cDNA Clones

[0136] Human SOM175 was also used to screen a mouse neonatal whole braincDNA library (Unizap, Stratagene). Four non-chimeric clones wereisolated: M175-A, B, C, D. All clones were partial cDNAs and M175-Ccontained several introns. Three of these cDNAs lacked the exon 6.

[0137] Another clone referred to as M1 was completely sequenced and wasfound to contain the full open reading frame plus part of the 5′utr andtotal 3′utr.

EXAMPLE 2 DNA Sequence Analysis

[0138] The entire sequence of the cDNA clone (SOM175) was compiled andis shown in FIG. 2 with its corresponding amino acid sequence. Thissequence was screened for open reading frames using the MAP program(GCG, University of Wisconsin). A single open reading frame of 672 bpwas observed (see FIG. 2). There appears to be little 5′ untranslatedsequences (2 bp). The 3′ untranslated region appears to be complete asit includes a poly-adenylation signal and poly-A tail.

[0139] Database homology searches were performed using the BLASTalgorithm (run at NCBI, USA). This analysis revealed homology to severalmammalian forms of VEGF (see FIG. 3). The amount of homology betweenSOM175 and human VEGF₁₆₅ was determined using the BESTFIT program (GCG,University of Wisconsin; see FIGS. 4 and 5). Nucleotide homology wasestimated at 69.7% and protein homology was estimated as at least 33.3%identity and 52.5% conservation using BESTFIT analysis. BLAST analysison nucleotide sequences revealed the almost complete match to a humanexpressed sequence tag EST06302 (Adams et al., 1993).

[0140] These data indicate that SOM175 encodes a growth factor that hasstructural similarities to VEGF. Both genes show start and stop codonsin similar positions and share discrete blocks of homology. All 8cysteines as well as a number of other VEGF residues believed to beinvolved in dimerisation are conserved. These residues are Cysteine-47,Proline-70, Cysteine-72, Valine-74, Arginine-77, Cysteine-78,Glycine-80, Cysteine-81, Cysteine-82, Cysteine-89, Proline-91,Cysteine-122 and Cysteine-124 and are shown in FIG. 6. Given thestructural conservation between VEGF and the SOM175 gene product it isalso possible that they share functional similarities. It is proposedthat SOM175 encodes a VEGF-like molecule that shares some propertieswith VEGF but has unique properties of its own. The nucleotide sequenceand corresponding amino acid sequence of VEGF₁₆₅ is shown in FIG. 1.

EXAMPLE 3

[0141] The percentage similarity and divergence between VEGF₁₆₅ familyand SOM175 family (protein) were analysed using the Clustal method,MegAlign Software, DNASTAR, Wisconsin. The results are shown in Tables2.1 and 2.2. The alternatively spliced forms of SOM175 are abbreviatedto SOM715-e6 where all of exon 6 is deleted; SOM715-e6 and 7 where allof exons 6 and 7 are deleted; and SOM175-e4 where all of exon 4 isdeleted. The spliced form of SOM175 are shown in FIG. 7. Genomic maps ofSOM175 showing intron/exon boundaries are shown in FIGS. 8a and 8 b.TABLE 2.1 VEGF₁₆₅ SOM175 SOM175-e6 SOM175-e6&7 SOM175-e4 A Percentnucleotide similarity between splice variants of SOM175 and humanVEGF₁₆₅ VEGF₁₆₅ *** 34.9 39.7 41.4 37.0 SOM175 *** 98.9 95.1 99.2SOM175-e6 *** 98.8 84.0 SOM175-e6&7 *** 80.3 SOM175-e4 *** B Percentnucleotide divergence between splice variants of SOM175 and humanVEGF₁₆₅ VEGF₁₆₅ *** 41.7 41.6 41.7 41.8 SOM175 *** 0.2 0.2 0.0 SOM175-e6*** 0.0 0.2 SOM175-e6&7 *** 0.3 SOM175-e4 ***

[0142] TABLE 2.2 A Percent amino acid identity between splice variantsof SOM175 and human VEGF₁₆₅ VEGF₁₆₅ SOM175 SOM175-e6 SOM175-e6&7SOM175-e4 VEGF₁₆₅ *** 31.4 42.3 33.5 40.6 SOM175 *** 74.7 73.7 99.1SOM175-e6 *** 76.8 99.1 SOM175-e6&7 *** 99.1 SOM175-e4 *** B Percentamino acid divergence between splice variants of SOM175 and humanVEGF₁₆₅ VEGF₁₆₅ *** 65.7 55.4 54.6 57.4 S0M175 *** 19.9 4.2 0.0SOM175-e6 *** 0.0 0.0 SOM175-e6&7 *** 0.0 SOM175-e4 ***

EXAMPLE 4 Bioassays to Determine Function of SOM175

[0143] Assays are conducted to evaluate whether SOM175 has similaractivities to VEGF on endothelial cell function, angiogenesis and woundhealing. Other assays are performed based on the results of receptorbinding distribution studies.

[0144] Assays of Endothelial Cell Function

[0145] Endothelial cell proliferation. Endothelial cell growth assays asdescribed in Ferrara & Henzel (1989) and in Gospodarowicz et al (1989).

[0146] Vascular permeability assay. This assay, which utilises the Milestest in guinea pigs, will be performed as described in Miles & Miles(1952).

[0147] Cell adhesion assay. The influence of SOM175 on adhesion ofpolymorphs to endothelial cells is analysed.

[0148] Chemotaxis. This is performed using the standard Boyden chamberchemotaxis assay.

[0149] Plasminogen activator assay. Endothelial cells are tested forplasminogen activator and plasminogen activator inhibitor productionupon addition of SOM175 (Pepper et al (1991)).

[0150] Endothelial cell migration assay. The ability of SOM175 tostimulate endothelial cells to migrate and form tubes is assayed asdescribed in Montesano et al (1986).

[0151] Angiogenesis Assay

[0152] SOM175 induction of an angiogenic response in chickchorioallantoic membrane is evaluated as described in Leung et al(1989).

[0153] Possible neurotrophic actions of SOM175 are assessed using thefollowing assays:

[0154] Neurite Outgrowth Assay and Gene Induction (PC12 Cells)

[0155] PC12 cells (a phaeochromocytoma cell line) respond to NGF andother neurotrophic factors by developing the characteristics ofsympathetic neurons, including the induction of early and late genes andthe extension of neurites. These cells are exposed to SOM175 and theirresponse monitored (Drinkwater et al (1991); and Drinkwater et al(1993)).

[0156] Cultured Neurons from the Peripheral Nervous System (PNS)

[0157] Primary cultures of the following PNS neurons are exposed toSOM175 and monitored for any response:

[0158] sensory neurons from neural crest and dorsal root ganglia

[0159] sympathetic neurons from sympathetic chain ganglia

[0160] placode derived sensory neurons from nodose ganglia

[0161] motoneurons from spinal cord

[0162] The assays are described in Suter et al (1992) and in Marinou etal (1992).

[0163] Where an in vitro response is observed, in vivo assays forproperties such as uptake and retrograde transport are performed asdescribed in Hendry et al (1992).

[0164] Nerve Regeneration (PNS)

[0165] Where neurotrophic effects of SOM175 are observed, its possiblerole in the regeneration of axotomised sensory neurons, sympatheticneurons and motoneurons is analysed by the methods of Otto et al (1989);Yip et al (1984) and Hendry et al (1976).

[0166] Actions of SOM175 on CNS Neurons

[0167] The ability of SOM175 to promote survival of central nervoussystem neurons is analysed as described in Hagg et al (1992); Williamset al (1986); Hefti (1986) and Kromer (1987).

[0168] Wound Healing

[0169] The ability of SOM175 to support wound healing are tested in themost clinically relevant model available, as described in Schilling etal (1959) and utilised by Hunt et al (1967).

[0170] The Haemopoietic System

[0171] A variety of in vitro and in vivo assays on specific cellpopulations of the haemopoietic system are available and are outlinedbelow:

[0172] Stem Cells

[0173] Murine

[0174] A variety of novel in vitro murine stem cell assays have beendeveloped using FACS-purified cells:

[0175] (a) Repopulating Stem Cells

[0176] These are cells capable of repopulating the bone marrow oflethally irradiated mice, and have the Lin⁻, Rh^(hi), Ly-6A/E⁺, c-kit⁺phenotype. The test substance is tested on these cells either alone, orby co-incubation with multiple factors, followed by measurement ofcellular proliferation by ³H thymidine incorporation.

[0177] (b) Late Stage Stem Cells

[0178] These are cells that have comparatively little bone marrowrepopulating ability but can generate D13 CFU-S. These cells have theLin⁻, Rh^(hi), Ly-6A/E⁺, c-kit⁺ phenotype. The test substance isincubated with these cells for a period of time, injected into lethallyirradiated recipients, and the number of D13 spleen colonies enumerated.

[0179] (c) Progenitor-Enriched Cells

[0180] These are cells that respond in vitro to single growth factors,and have the Lin⁻, Rh^(hi), Ly-6A/E⁺, c-kit⁺ phenotype. This assay willshow if SOM175 can act directly on haemopoietic progenitor cells. Thetest substance is incubated with these cells in agar cultures, and thenumber of colonies enumerated after 7-14 days.

[0181] Atherosclerosis

[0182] Smooth muscle cells play a crucial role in the development orinitiation of atherosclerosis, requiring a change in their phenotypefrom a contractile to a synthetic state. Macrophages, endothelial cells,T lymphocytes and platelets all play a role in the development ofatherosclerotic plaques by influencing the growth and phenotypicmodulations of smooth muscle cell. An in vitro assay that measures theproliferative rate and phenotypic modulations of smooth muscle cells ina multicellular environment is used to assess the effect of SOM175 onsmooth muscle cells. The system uses a modified Rose chamber in whichdifferent cell types are seeded onto opposite coverslips.

[0183] Effects of SOM175 on Bone

[0184] The ability of SOM175 to regulate proliferation of osteoblasts isassayed as described in Lowe et al (1991). Any effects on boneresorption are assayed as described in Lowe et al (1991). Effects onosteoblast migration and changes in intracellular molecules (e.g. cAMPaccumulation, alkaline phosphatase levels) are analysed as described inMidy et al (1994).

[0185] Effects on Skeletal Muscle Cells

[0186] Effects of SOM175 on proliferation of myoblasts and developmentof myotubes can be determined as described by Ewton et al (1980) and byGospodarowicz et al (1976).

EXAMPLE 5 Cloning Murine VEGF DNA

[0187] Isolation of cDNAs

[0188] Murine VRF (mVRF) clones were selected from a lambda Zap new bornwhole brain cDNA library (Stratagene). Primary phage from high densityfilters (5×10⁴ pfu/plate) were identified by hybridisation with a 682 bp³²P-labelled probe generated by PCR from an hVRF cDNA (pSOM175) asdescribed above. Hybridisation and stringent washes of nylon membranes(Hybond-N) were carried out at 65° C. under conditions described byChurch and Gilbert (1984). Positive plaques were picked, purified andexcised in vivo to produce bacterial colonies containing cDNA clones inpbluescript SK-.

[0189] Isolation of Genomic Clones

[0190] Genomic clones were isolated from a mouse strain SV/129 librarycloned in the lambda Fix II vector (Stratagene). High density filters(5×10⁴ pfu/filter) were screened with a 563 bp ³²P-labelled probegenerated by PCR amplification of the nucleotide 233-798 region of themVRF cDNA (see FIG. 9). Positive clones were plugged and re-screenedwith filters containing 400-800 pfu. Large scale phage preparations wereprepared using the QIAGEN lambda kit or by ZnCl₂ purification (Santos,1991).

[0191] Nucleotide Sequencing and Analysis

[0192] cDNAs were sequenced on both strands using a variety ofvector-based and internal primers with Applied Biosystems Incorporated(ABI) dye terminator sequencing kits according to the manufacturer'sspecifications. Sequences were analysed on an ABI Model 373A automatedDNA sequencer. Peptide homology alignments were performed using theprogram BESTFIT (GCG, Wisconsin).

[0193] Identification of Intron/Exon Boundaries

[0194] Identification of exon boundaries and flanking regions wascarried out using PCR with mouse genomic DNA or mVRF genomic lambdaclones as templates. The primers used in PCR to identify introns werederived from the hVRF sequence and to allow for potential human-mousesequence mismatches annealing temperatures 5-10° C. below the estimatedT_(m) were used. All PCR products were sized by agarose gelelectrophoresis and gel purified using QIAquick spin columns (Qiagen)and the intron/exon boundaries were sequenced directly from theseproducts. In addition, some splice junctions were sequenced fromsubcloned genomic fragments of MVRF. Intron/exon boundaries wereidentified by comparing cDNA and genomic DNA sequences.

[0195] Northern Analysis

[0196] Total cellular RNA was prepared from a panel of fresh normaladult mouse tisues (brain, kidney, liver, muscle) using the method ofChomczynski and Sacchi (1987). 20 μg of total RNA were electrophoresed,transferred to a nylon membrane (Hybond N, Amersham) and hybridisedunder standard conditions (Church & Gilbert, 1984). Filters were washedat 65° C. in 0.1×SSC (20×SSC is 3M NaCl/0.3M trisodium citrate), 0.1%SDS and exposed to X-ray film with intensifying screens at −70° C. for1-3 days.

[0197] Characterisation of mVRF cDNAs

[0198] Murine VRF homologues were isolated by screening a murine cDNAlibrary with an hVRF cDNA clone. Five clones of sizes varying from0.8-1.5 kb were recovered and sequenced. The cDNA sequences werecomplied to give a full length 1041 bp cDNA sequence covering the entireopen reading frame (621 bp or 564 bp depending on the splice form, seebelow) and 3′ UTR (379 bp), as well as 163 bp of the 5′ UTR (FIG. 9).

[0199] The predicted initiation codon matched the position of the startcodon in hVRF. One other out of frame ATG was located at position −47and two termination codons were observed upstream positions −9 and −33,respectively) and in frame with the putative initiation codon.

[0200] The predicted N-terminal signal peptide of hVRF appears to bepresent in mVRF with 81% identity (17/21 amino acids). Peptide cleavagewithin mVRF is expected to occur after reside 21 (FIG. 10). These datasuggest that mature mVRF is secreted and could therefore conceivablyfunction as a growth factor.

[0201] As with hVRF, two open reading frames (ORFs) were detected incDNAs isolated by library screening. Four of five clones were found tobe alternatively spliced and lacked a 101 bp fragment homologous to exon6 of hVRF. The predicted peptide sequences of the two isoforms of mVRFwere determined and aligned with the corresponding human isoforms (FIG.10).

[0202] The message encoding mVRF₁₈₆ contains a 621 bp ORF with codingsequences terminating at position +622, towards the end of exon 7 (FIG.9). The smaller message encoding mVRF₁₆₇ actually terminates downstreamof the +622 TAG site due to a frame shift resulting from splicing out ofthe 101 bp exon 6 and the introduction of a stop codon (TGA) at position+666, near the beginning of exon 8 (FIG. 9).

[0203] The mVRF₁₈₆ protein has strong homology to the amino and centralportions of VEGF while the carboxyl end is completely divergent an isalanine rich. mVRF₁₆₇ possesses these similarities and also maintainshomology to mVEGF right through to the C-terminus (FIG. 11). The overallhomology of mVRF₁₆₇ to hVRF₁₆₇ was 85% identity and 92% similarity,respectively (FIG. 10). Likewise, homology between mVRF₁₆₇ and mVEGF(Breier et al, 1992) was 49% identity and 71% conservative amino acidsubstitution, respectively (FIG. 11).

[0204] A canonical vertebrate polyadenylation signal (AATAAA) (Birnstielet al, 1986) was not present in the mVRF cDNA, however, the closelymatching sequence GATAAA is present at similar positions in both mouseand human VRF cDNAs (FIG. 9). In contrast to hVRF, mVRF was found tocontain an AC dinucleotide repeat at the extreme 3′ end of the 3′ UTR(nucleotide positions 998 to 1011, FIG. 9). Polymorphism of this repeatregion was observed between some of the mVRF cDNAs, with the number ofdinucleotides varying from 7 to 11.

[0205] Genomic Characterisation of mVRF

[0206] Intron/exon boundaries (Table 3) were mapped using primers whichflanked sequences homologous to the corresponding hVRF boundaries.Introns I, III, IV and VI of mVRF (Table 3, FIG. 12) were smaller thanthe hVRF intervening sequences. The complete genomic sequence wascompiled from the 5′ UTR of mVRF through to intron VI, the largestintervening region (2.2 kb), by sequencing amplified introns and clonedgenomic portions of mVRF. There was only one major difference in genomicstructure between mVRF and hVRF and that was the exon 7/intron VIboundary of mVRF was located 10 bp further downstream in relation to thecDNA sequence, hence exon 7 in mVRF is 10 bp longer than thecorresponding exon in hVRF.

[0207] Exons 6 and 7 are contiguous in mVRF, as has been found to occurin the human homologue. The strong sequence homology between exon 6 ofmVRF and hVRF (FIG. 10) suggests that this sequence is not a retainedintronic sequence but rather encodes a functional part of the VRF₁₈₆isoform.

[0208] General intron/exon structure is conserved between the variousmembers of the VEGF gene family (VEGF, PIGF, hVRF) and therefore it isnot surprising that the overall genomic organisation of the mVRF gene isvery similar to these genes (FIG. 12).

[0209] Previous comparative mapping studies have shown that the regionsurrounding the human multiple endocrine neoplasia type 1 disease locuson chromosome 11q13 is syntenic with the proximal segment of mousechromosome 19 (Rochelle et al, 1992). Since the inventors have mappedthe hVRF gene to within 1 kb of the human MEN1 locus (see above) it ismost likely that the murine VRF gene maps near the centromere ofchromosome 19.

[0210] Expression Studies of mVRF

[0211] Northern analysis of RNA from adult mouse tissues (muscle, heart,lung and liver) showed that expression appears to be ubiquitous andoccurs primarily as a major band of approximately 1.3 kb in size (FIG.14). This is somewhat different to the pattern observed for hVRF inwhich two major bands of 2.0 and 5.5 kb have been identified in alltissues examined. The 1.3 kb murine message presumably corresponds tothe shorter of the human transcripts and the size variation thereof ismost likely due to a difference in the length of the respective 5′ UTRs.

EXAMPLE 6 Expression of Murine VEGF in Pre- and Post-Natal Mouse

[0212] Animals

[0213] Timed pregnant (n=4) and young adult (n=2) mice (C57 inbredstrain, ALAB, Sweden) were sacrificed with carbon dioxide, and therelevant tissues were taken out and frozen on a chuck. Tissues were keptat −70° C. until further use. Two gestational ages was used in thisstudy; embryonic day 8 (E8), 14 and E17.

[0214] In situ Hybridisation Histochemistry

[0215] In situ hybridisation was performed as previously described(Dagerlind et al, 1992). Briefly, transverse sections (14 μm) were cutin a cryostat (Microm, Germany), thawed onto Probe-On slides (FisherScientific, USA) and stored in black sealed boxes at −70° C. until used.The sequences of the synthetic 42-mer oligonucleotides complementary tomRNA encoding mVRF were ACCACCACCTCCCTGGGCTGGCATGTGGCACGTGCATAAACG [SEQID NO:11] (complementary to nt 120-161) andAGTTGTTTGACCACATTGCCCATGAGTTCCATGCTCAGAGGC [SEQ ID NO:12] (complementaryto nt 162-203). To detect the two alternative splice formsoligonucleotide GATCCTGGGGCTGGAGTGGGATGGATGATGTCAGCTGG [SEQ ID NO:13](complementary to nt xxx-xxx) andGCGGGCAGAGGATCCTGGGGCTGTCTGGCCTCACAGCACT [SEQ ID NO:14] were used. Theprobes were labeled at the 3′-end withdeoxyadenosine-alpha[thio]triphosphate [³⁵S] (NEN, USA) using terminaldeoxynucleotidyl transferase (IBI, USA) to a specific activity of7-10×10⁸ cpm/μg and hybridised to the sections without pretreatment for16-18 h at 42° C. The hybridisation mixture contained: 50% v/vformamide, 4×SSC (1×SSC=0.15M NaCl and 0.015M sodium-citrate),1×Denhardt's solution (0.02% each of polyvinyl-pyrrolidone, BSA andFicoll) 1% v/v sarcosyl (N-lauroylsarcosine; Sigma), 0.02M phosphatebuffer (pH 7.0), 10% w/v dextran sulfate (Pharmacia, Sweden), 250 μg/mlyeast tRNA (Sigma), 500 μg/ml sheared and heat denatured salmon spermDNA (Sigma) and 200 mM dithiothreitol (DTT; LKB, Sweden). In controlsections, the specificity of both probes was checked by adding a 20-foldexcess of unlabeled probe to the hybridisation mixture. In addition,adjacent sections were hybridised with a probe unrelated to this studywhich gave a different expression pattern. Following hybridisation thesections were washed several times in 1×SSC at 55° C., dehydrated inethanol and dipped in NTB2 nuclear track emulsion (Kodak, USA). After3-5 weeks the sections were development in D-19 developer (Kodak, USA)and cover-slipped. In some cases, sections were opposed to anautoradiographic film (Beta-max autoradiography film Amersham Ltd, UK)prior to emulsion-dipping.

[0216] The four different probes gave identical hybridisation patternsin all tissues examined. Mouse VRF expression was detecting already inthe E8 embryo, in which positive signal was recorded over structuresmost likely corresponding to the neuronal tube. In sagittal sections ofE14 mouse embryo the strongest hybridisation signal was present overheart and in the nervous system, especially cerebral cortex (FIG. 14A).A low level of expression was present in all other tissues. At a latergestational age, E17, a high mVRF mRNA signal was confined to he heartand brown fat tissue in the back and around the neck (FIG. 14B). Clearlypositive hybridisation signals were present in the gray of the spinalcord and in the tongue (FIG. 14B). Expression in the cerebral cortex wasclearly reduced compared to day 14. The weak background expression seenin the E14 embryo in for example muscle, had decreased at thisgestational age. A strong mVRF mRNA hybridisation signal was presentsolely over the heart and in the brown fat in the young adult mice (FIG.14C). The signal over the heart was evenly distributed ove the entireventricular wall, including the papillary muscles (FIG. 14D). Insections of heart tissue hybridised with an excess of cold probe, nospecific labeling over background signal was recorded (FIG. 14E).

[0217] Apart from the heart, mVRF mRNA signal was present over certaintissues on the outside of the thoracic cage that morphologicallyresembled brown fat. This was verified with sudan black counterstaining,which showed a strong staining in the same areas (FIGS. 15A and 15B). Intransverse sections of adult mouse spinal cord, the mVRF probes gave aneuronal staining pattern over the gray matter (FIG. 15C).Counterstaining with toluidine (FIG. 15D) showed that motoneurons in theventral horn (FIGS. 15C and 15D), interneurons (FIG. 15C) in the deeppart of the dorsal horn and around the central canal where to a largeextent positive for mVRF mRNA.

EXAMPLE 7 Effects of VEGF and SOM175 Proteins on Chick Sensory Neurons

[0218] The effects of VEGF and SOM175 proteins on embryonic day 8 chicksensory neurons were determined using the method of Nurcombe et al(1992). The neuronal assay was read at 48 hours using 2000 cells perassay well. The results were obtained using ³H-thymidine counts. Thepercentage survival of neurons, neurite outgrowth and average neuritelength in μm were determined using NGF as positive control and variousconcentrations of VEGF, VEGF in the presence of heparin and VEGF in thepresence of heparin and 5 μM, 5′-flurouracil (5FU). 5FU kills glialcells.

[0219] The results are shown in FIG. 16. The results show that VEGF iseffective in promoting neuronal survival but that this requires thepresence of glial cells. FIG. 17 shows the results of the effect of VEGFand SOM175 on three types of chick glia. The glia tested were CNS glia,peripheral glia and CNS oligodendrocytes. Heparin was used as 10 μg/mlin all cultures and the assay was read at 24 hours. Results weremeasured in ³H-thymidine counts using 2000 cells per well.

[0220] The results show that for chick central and peripheral neurons,astroglia were markedly stimulated to proliferate by SOM175 in thepresence of heparin but that chick oligodendrocytes showed negligibleincrease in the rate of division.

EXAMPLE 8 Effects of SOM175 Proteins on Mouse Primary and CentralNeurons

[0221] The results in Example 7 show that VEGF isoform had an effect onchick primary and central neurons through the agency of the astroglialcells. Similar experiments were repeated in mouse cells.

[0222] Culture Conditions

[0223] Neuronal and gligal cells for all in vitro experiments wereprepared and cultured according o the techniques described in “Methodsin Neurosciences (Vol. 2): Cell Culture” Ed. P. M. Conn, Academic Press,San Diego, 1990, pp33-46 for astroglial cells, pp56-74 foroligodendroglial cells, and pp87-102 for central neurons.

[0224] Cells were plated onto 24-well culture clusters (Nunc) coatedwith poly-L-ornithine (0.1 mg/ml, 1 h) at a density of 2,000 cells/well.After 48 hours in culture, neurons were counted in the wells underinverted phase light using well established techniques (Maruta et al.1993) and glial cells assessed with [³H]thymidine uptake to monitor celldivision rates as below. Heparin (10 μg/ml, low molecular weightfraction, Sigma Chemical Corp.) was present at all times in the culturemedia except where noted. The neuronal cultures were supplemented with 5mM 5-fluoro-2-deoxyuridine (Sigma) to suppress background glial growth.

[0225]³H-Thymidine Incorporation Assay for Glial Cell Proliferation

[0226] The cells were pulsed for 14 h with ³H-thymidine (specificactivity 103 μCi/ug) fraom a stock concentration of 0.1 mCi/ml instandard medium, giving a final incubating volume of 20 μl/well. Thecontents of the wells were harvested and absorbed onto nitrocellulosepaper (Titertek, Flow). Remaining adherent cells were removed byincubation with trypsin/versene (CSL Limited, Victoria, Australia) for 5min. This procedure was carried out twice. The nitrocellulose discs werewashed in a standard Titertek harvester (Flow) using first distilledwater, and then methanol. The nitrocellulose discs were dried,scintillation fluid (containing 5% v/v Triton-X) added and the discscounted on a scintillation counter.

[0227] Greatest activity was seen with preparations of SOM175 absentexon 6 (SOMΔX6) on mouse astroglial cell cultures, where there was asignificant stimulus to their proliferation when delivered inconjunction with heparin (FIG. 16). Little stimulus was given to theproliferation of oligodendroglial cells (FIG. 17), and very littlediscernable potentiation of the survival response of isolated forebrainneurons (FIG. 18). The standard deviation on all three graphs for eachpoint was less than 8%.

[0228] The viability of neurons can be maintained by promoting glialcell proliferation. Furthermore, SOMΔX6 is a good inducer of astroglialproliferation and may be expressed in conjunction with the formation ofastroglial endfeet on central nervous system endothelial cells.

[0229] Those skilled in the art will appreciate that the inventiondescribed herein is susceptible to variations and modifications otherthan those specifically described. It is to be understood that theinvention includes all such variations and modifications. The inventionalso includes all of the steps, features, compositions and compoundsreferred to or indicated in this specification, individually orcollectively, and any and all combinations of any two or more of saidsteps or features. TABLE 3 Splice junctions of the murine VRF gene 5′UTR* . . . Exon 1 >223 bp CCCAGgtacgtgcgt Intron I 495 bpttccccacagGCCCC Exon 2 43 bp GAAAGgtaataatag Intron II 288 bpctgcccacagTGGTG Exon 3 197 bp TGCAGgtaccagggc Intron III 196 bpctgagcacagATCCT Exon 4 74 bp TGCAGgtgccagccc Intron IV 182 bpctcttttcagACCTA Exon 5 36 bp GACAGattcttggtg Intron V 191 bpctcctcctagGGTTG Exon 6 101 bp (no intron) CCCACTCCAGCCCCA Exon 7 135 bpTGTAGgtaaggagtc Intron VI ˜2200 bp cactccccagGTGCC Exon 8 394 bpAGAGATGGAGACACT

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1 22 1 649 DNA Nucleotide Sequence of VEGF165 CDS (17)..(589) 1tcgggcctcc gaaacc atg aac ttt ctg ctg tct tgg gtg cat tgg agc ctt 52 MetAsn Phe Leu Leu Ser Trp Val His Trp Ser Leu 1 5 10 gcc ttg ctg ctc tacctc cac cat gcc aag tgg tcc cag gct gca ccc 100 Ala Leu Leu Leu Tyr LeuHis His Ala Lys Trp Ser Gln Ala Ala Pro 15 20 25 atg gca gaa gga gga gggcag aat cat cac gaa gtg gtg aag ttc atg 148 Met Ala Glu Gly Gly Gly GlnAsn His His Glu Val Val Lys Phe Met 30 35 40 gat gtc tat cag cgc agc tactgc cat cca atc gag acc ctg gtg gac 196 Asp Val Tyr Gln Arg Ser Tyr CysHis Pro Ile Glu Thr Leu Val Asp 45 50 55 60 atc ttc cag gag tac cct gatgag atc gag tac atc ttc aag cca tcc 244 Ile Phe Gln Glu Tyr Pro Asp GluIle Glu Tyr Ile Phe Lys Pro Ser 65 70 75 tgt gtg ccc ctg atg cga tgc gggggc tgc tgc aat gac gag ggc ctg 292 Cys Val Pro Leu Met Arg Cys Gly GlyCys Cys Asn Asp Glu Gly Leu 80 85 90 gag tgt gtg ccc act gag gag tcc aacatc acc atg cag att atg cgg 340 Glu Cys Val Pro Thr Glu Glu Ser Asn IleThr Met Gln Ile Met Arg 95 100 105 atc aaa cct cac caa ggc cag cac atagga gag atg agc ttc cta cag 388 Ile Lys Pro His Gln Gly Gln His Ile GlyGlu Met Ser Phe Leu Gln 110 115 120 cac aac aaa tgt gaa tgc aga cca aagaaa gat aga gca aga caa gaa 436 His Asn Lys Cys Glu Cys Arg Pro Lys LysAsp Arg Ala Arg Gln Glu 125 130 135 140 aat ccc tgt ggg cct tgc tca gagcgg aga aag cat ttg ttt gta caa 484 Asn Pro Cys Gly Pro Cys Ser Glu ArgArg Lys His Leu Phe Val Gln 145 150 155 gat ccg cag acg tgt aaa tgt tcctgc aaa aac aca gac tcg cgt tgc 532 Asp Pro Gln Thr Cys Lys Cys Ser CysLys Asn Thr Asp Ser Arg Cys 160 165 170 aag gcg agg cag ctt gag tta aacgaa cgt act tgc aga tgt gac aag 580 Lys Ala Arg Gln Leu Glu Leu Asn GluArg Thr Cys Arg Cys Asp Lys 175 180 185 ccg agg cgg tgagccgggcaggaggaagg agcctccctc agcgtttcgg 629 Pro Arg Arg 190 gaaccagatctctcaccagg 649 2 191 PRT Nucleotide Sequence of VEGF165 2 Met Asn PheLeu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr LeuHis His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly 20 25 30 Gly GlyGln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln 35 40 45 Arg SerTyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu 50 55 60 Tyr ProAsp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu 65 70 75 80 MetArg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro 85 90 95 ThrGlu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His 100 105 110Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys 115 120125 Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly 130135 140 Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr145 150 155 160 Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys AlaArg Gln 165 170 175 Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys ProArg Arg 180 185 190 3 1094 DNA Nucleotide Sequence of SOM175 CDS(3)..(623) 3 cc atg agc cct ctg ctc cgc cgc ctg ctg ctc gcc gca ctc ctgcag 47 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln 1 510 15 ctg gcc ccc gcc cag gcc cct gtc tcc cag cct gat gcc cct ggc cac 95Leu Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His 20 25 30cag agg aaa gtg gtg tca tgg ata gat gtg tat act cgc gct acc tgc 143 GlnArg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys 35 40 45 cagccc cgg gag gtg gtg gtg ccc ttg act gtg gag ctc atg ggc acc 191 Gln ProArg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr 50 55 60 gtg gccaaa cag ctg gtg ccc agc tgc gtg act gtg cag cgc tgt ggt 239 Val Ala LysGln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly 65 70 75 ggc tgc tgccct gac gat ggc ctg gag tgt gtg ccc act ggg cag cac 287 Gly Cys Cys ProAsp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His 80 85 90 95 caa gtc cggatg cag atc ctc atg atc cgg tac ccg agc agt cag ctg 335 Gln Val Arg MetGln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu 100 105 110 ggg gag atgtcc ctg gaa gaa cac agc cag tgt gaa tgc aga cct aaa 383 Gly Glu Met SerLeu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys 115 120 125 aaa aag gacagt gct gtg aag cca gac agg gct gcc act ccc cac cac 431 Lys Lys Asp SerAla Val Lys Pro Asp Arg Ala Ala Thr Pro His His 130 135 140 cgt ccc cagccc cgt tct gtt ccg ggc tgg gac tct gcc ccc gga gca 479 Arg Pro Gln ProArg Ser Val Pro Gly Trp Asp Ser Ala Pro Gly Ala 145 150 155 ccc tcc ccagct gac atc acc cat ccc act cca gcc cca ggc ccc tct 527 Pro Ser Pro AlaAsp Ile Thr His Pro Thr Pro Ala Pro Gly Pro Ser 160 165 170 175 gcc cacgct gca ccc agc acc acc agc gcc ctg acc ccc gga cct gcc 575 Ala His AlaAla Pro Ser Thr Thr Ser Ala Leu Thr Pro Gly Pro Ala 180 185 190 gct gccgct gcc gac gcc gca gct tcc tcc gtt gcc aag ggc ggg gct 623 Ala Ala AlaAla Asp Ala Ala Ala Ser Ser Val Ala Lys Gly Gly Ala 195 200 205tagagctcaa cccagacacc tgcaggtgcc ggaagctgcg aaggtgacac atggcttttc 683agactcagca gggtgacttg cctcagaggc tatatcccag tgggggaaca aaggggagcc 743tggtaaaaaa cagccaagcc cccaagacct cagcccaggc agaagctgct ctaggacctg 803ggcctctcag agggctcttc tgccatccct tgtctccctg aggccatcat caaacaggac 863agagttggaa gaggagactg ggaggcagca agaggggtca cataccagct caggggagaa 923tggagtactg tctcagtttc taaccactct gtgcaagtaa gcatcttaca actggctctt 983cctcccctca ctaagaagac ccaaacctct gcataatggg atttgggctt tggtacaaga 1043actgtgaccc ccaaccctga taaaagagat ggaaggaaaa aaaaaaaaaa a 1094 4 207 PRTNucleotide Sequence of SOM175 4 Met Ser Pro Leu Leu Arg Arg Leu Leu LeuAla Ala Leu Leu Gln Leu 1 5 10 15 Ala Pro Ala Gln Ala Pro Val Ser GlnPro Asp Ala Pro Gly His Gln 20 25 30 Arg Lys Val Val Ser Trp Ile Asp ValTyr Thr Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu Val Val Val Pro Leu ThrVal Glu Leu Met Gly Thr Val 50 55 60 Ala Lys Gln Leu Val Pro Ser Cys ValThr Val Gln Arg Cys Gly Gly 65 70 75 80 Cys Cys Pro Asp Asp Gly Leu GluCys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg Met Gln Ile Leu Met IleArg Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu Met Ser Leu Glu Glu HisSer Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 Lys Asp Ser Ala Val LysPro Asp Arg Ala Ala Thr Pro His His Arg 130 135 140 Pro Gln Pro Arg SerVal Pro Gly Trp Asp Ser Ala Pro Gly Ala Pro 145 150 155 160 Ser Pro AlaAsp Ile Thr His Pro Thr Pro Ala Pro Gly Pro Ser Ala 165 170 175 His AlaAla Pro Ser Thr Thr Ser Ala Leu Thr Pro Gly Pro Ala Ala 180 185 190 AlaAla Ala Asp Ala Ala Ala Ser Ser Val Ala Lys Gly Gly Ala 195 200 205 5993 DNA Nuc. Seq. of SOM175 Absent Exon 6 CDS (3)..(566) 5 cc atg agccct ctg ctc cgc cgc ctg ctg ctc gcc gca ctc ctg cag 47 Met Ser Pro LeuLeu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln 1 5 10 15 ctg gcc ccc gcccag gcc cct gtc tcc cag cct gat gcc cct ggc cac 95 Leu Ala Pro Ala GlnAla Pro Val Ser Gln Pro Asp Ala Pro Gly His 20 25 30 cag agg aaa gtg gtgtca tgg ata gat gtg tat act cgc gct acc tgc 143 Gln Arg Lys Val Val SerTrp Ile Asp Val Tyr Thr Arg Ala Thr Cys 35 40 45 cag ccc cgg gag gtg gtggtg ccc ttg act gtg gag ctc atg ggc acc 191 Gln Pro Arg Glu Val Val ValPro Leu Thr Val Glu Leu Met Gly Thr 50 55 60 gtg gcc aaa cag ctg gtg cccagc tgc gtg act gtg cag cgc tgt ggt 239 Val Ala Lys Gln Leu Val Pro SerCys Val Thr Val Gln Arg Cys Gly 65 70 75 ggc tgc tgc cct gac gat ggc ctggag tgt gtg ccc act ggg cag cac 287 Gly Cys Cys Pro Asp Asp Gly Leu GluCys Val Pro Thr Gly Gln His 80 85 90 95 caa gtc cgg atg cag atc ctc atgatc cgg tac ccg agc agt cag ctg 335 Gln Val Arg Met Gln Ile Leu Met IleArg Tyr Pro Ser Ser Gln Leu 100 105 110 ggg gag atg tcc ctg gaa gaa cacagc cag tgt gaa tgc aga cct aaa 383 Gly Glu Met Ser Leu Glu Glu His SerGln Cys Glu Cys Arg Pro Lys 115 120 125 aaa aag gac agt gct gtg aag ccagat agc ccc agg ccc ctc tgc cca 431 Lys Lys Asp Ser Ala Val Lys Pro AspSer Pro Arg Pro Leu Cys Pro 130 135 140 cgc tgc acc cag cac cac cag cgccct gac ccc cgg acc tgc cgc tgc 479 Arg Cys Thr Gln His His Gln Arg ProAsp Pro Arg Thr Cys Arg Cys 145 150 155 cgc tgc cga cgc cgc agc ttc ctccgt tgc caa ggg cgg ggc tta gag 527 Arg Cys Arg Arg Arg Ser Phe Leu ArgCys Gln Gly Arg Gly Leu Glu 160 165 170 175 ctc aac cca gac acc tgc aggtgc cgg aag ctg cga agg tgacacatgg 576 Leu Asn Pro Asp Thr Cys Arg CysArg Lys Leu Arg Arg 180 185 cttttcagac tcagcagggt gacttgcctc agaggctatatcccagtggg ggaacaaagg 636 ggagcctggt aaaaaacagc caagccccca agacctcagcccaggcagaa gctgctctag 696 gacctgggcc tctcagaggg ctcttctgcc atcccttgtctccctgaggc catcatcaaa 756 caggacagag ttggaagagg agactgggag gcagcaagaggggtcacata ccagctcagg 816 ggagaatgga gtactgtctc agtttctaac cactctgtgcaagtaagcat cttacaactg 876 gctcttcctc ccctcactaa gaagacccaa acctctgcataatgggattt gggctttggt 936 acaagaactg tgacccccaa ccctgataaa agagatggaaggaaaaaaaa aaaaaaa 993 6 188 PRT Nuc. Seq. of SOM175 Absent Exon 6 6 MetSer Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln Leu 1 5 10 15Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His Gln 20 25 30Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 35 40 45Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr Val 50 55 60Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 65 70 7580 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 9095 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 100105 110 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys115 120 125 Lys Asp Ser Ala Val Lys Pro Asp Ser Pro Arg Pro Leu Cys ProArg 130 135 140 Cys Thr Gln His His Gln Arg Pro Asp Pro Arg Thr Cys ArgCys Arg 145 150 155 160 Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly ArgGly Leu Glu Leu 165 170 175 Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu ArgArg 180 185 7 858 DNA Nuc. Seq. of SOM175 Absent Exons 6&7 CDS(3)..(431) 7 cc atg agc cct ctg ctc cgc cgc ctg ctg ctc gcc gca ctc ctgcag 47 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln 1 510 15 ctg gcc ccc gcc cag gcc cct gtc tcc cag cct gat gcc cct ggc cac 95Leu Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro Gly His 20 25 30cag agg aaa gtg gtg tca tgg ata gat gtg tat act cgc gct acc tgc 143 GlnArg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala Thr Cys 35 40 45 cagccc cgg gag gtg gtg gtg ccc ttg act gtg gag ctc atg ggc acc 191 Gln ProArg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr 50 55 60 gtg gccaaa cag ctg gtg ccc agc tgc gtg act gtg cag cgc tgt ggt 239 Val Ala LysGln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly 65 70 75 ggc tgc tgccct gac gat ggc ctg gag tgt gtg ccc act ggg cag cac 287 Gly Cys Cys ProAsp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His 80 85 90 95 caa gtc cggatg cag atc ctc atg atc cgg tac ccg agc agt cag ctg 335 Gln Val Arg MetGln Ile Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu 100 105 110 ggg gag atgtcc ctg gaa gaa cac agc cag tgt gaa tgc aga cct aaa 383 Gly Glu Met SerLeu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys 115 120 125 aaa aag gacagt gct gtg aag cca gat agg tgc cgg aag ctg cga agg 431 Lys Lys Asp SerAla Val Lys Pro Asp Arg Cys Arg Lys Leu Arg Arg 130 135 140 tgacacatggcttttcagac tcagcagggt gacttgcctc agaggctata tcccagtggg 491 ggaacaaaggggagcctggt aaaaaacagc caagccccca agacctcagc ccaggcagaa 551 gctgctctaggacctgggcc tctcagaggg ctcttctgcc atcccttgtc tccctgaggc 611 catcatcaaacaggacagag ttggaagagg agactgggag gcagcaagag gggtcacata 671 ccagctcaggggagaatgga gtactgtctc agtttctaac cactctgtgc aagtaagcat 731 cttacaactggctcttcctc ccctcactaa gaagacccaa acctctgcat aatgggattt 791 gggctttggtacaagaactg tgacccccaa ccctgataaa agagatggaa ggaaaaaaaa 851 aaaaaaa 858 8143 PRT Nuc. Seq. of SOM175 Absent Exons 6&7 8 Met Ser Pro Leu Leu ArgArg Leu Leu Leu Ala Ala Leu Leu Gln Leu 1 5 10 15 Ala Pro Ala Gln AlaPro Val Ser Gln Pro Asp Ala Pro Gly His Gln 20 25 30 Arg Lys Val Val SerTrp Ile Asp Val Tyr Thr Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu Val ValVal Pro Leu Thr Val Glu Leu Met Gly Thr Val 50 55 60 Ala Lys Gln Leu ValPro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 65 70 75 80 Cys Cys Pro AspAsp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg Met GlnIle Leu Met Ile Arg Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu Met SerLeu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 Lys AspSer Ala Val Lys Pro Asp Arg Cys Arg Lys Leu Arg Arg 130 135 140 9 910DNA Nuc. Seq. of SOM175 Absent Exon 4 CDS (3)..(305) 9 cc atg agc cctctg ctc cgc cgc ctg ctg ctc gcc gca ctc ctg cag 47 Met Ser Pro Leu LeuArg Arg Leu Leu Leu Ala Ala Leu Leu Gln 1 5 10 15 ctg gcc ccc gcc caggcc cct gtc tcc cag cct gat gcc cct ggc cac 95 Leu Ala Pro Ala Gln AlaPro Val Ser Gln Pro Asp Ala Pro Gly His 20 25 30 cag agg aaa gtg gtg tcatgg ata gat gtg tat act cgc gct acc tgc 143 Gln Arg Lys Val Val Ser TrpIle Asp Val Tyr Thr Arg Ala Thr Cys 35 40 45 cag ccc cgg gag gtg gtg gtgccc ttg act gtg gag ctc atg ggc acc 191 Gln Pro Arg Glu Val Val Val ProLeu Thr Val Glu Leu Met Gly Thr 50 55 60 gtg gcc aaa cag ctg gtg ccc agctgc gtg act gtg cag cgc tgt ggt 239 Val Ala Lys Gln Leu Val Pro Ser CysVal Thr Val Gln Arg Cys Gly 65 70 75 ggc tgc tgc cct gac gat ggc ctg gagtgt gtg ccc act ggg cag cac 287 Gly Cys Cys Pro Asp Asp Gly Leu Glu CysVal Pro Thr Gly Gln His 80 85 90 95 caa gtc cgg atg cag acc taaaaaaaaggacagtgctg tgaagccaga 335 Gln Val Arg Met Gln Thr 100 cagggctgccactccccacc accgtcccca gccccgttct gttccgggct gggactctgc 395 ccccggagcaccctccccag ctgacatcac ccatcccact ccagccccag gcccctctgc 455 ccacgctgcacccagcacca ccagcgccct gacccccgga cctgccgctg ccgctgccga 515 cgccgcagcttcctccgttg ccaagggcgg ggcttagagc tcaacccaga cacctgcagg 575 tgccggaagctgcgaaggtg acacatggct tttcagactc agcagggtga cttgcctcag 635 aggctatatcccagtgggga acaaagagga gcctggtaaa aaacagccaa gcccccaaga 695 cctcagcccaggcagaagct gctctaggac ctgggcctct cagagggctc ttctgccatc 755 ccttgtctccctgaggccat catcaaacag gacagagttg gaagaggaga ctgggaggca 815 gcaagaggggtcacatacca gctcagggga gaatggagta ctgtctcagt ttctaaccac 875 tctgtgcaagtaagcatctt acaactggct cttcc 910 10 101 PRT Nuc. Seq. of SOM175 AbsentExon 4 10 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu GlnLeu 1 5 10 15 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro GlyHis Gln 20 25 30 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala ThrCys Gln 35 40 45 Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met GlyThr Val 50 55 60 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg CysGly Gly 65 70 75 80 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr GlyGln His Gln 85 90 95 Val Arg Met Gln Thr 100 11 42 DNA Oligonucleotide11 accaccacct ccctgggctg gcatgtggca cgtgcataaa cg 42 12 42 DNAOligonucleotide 12 agttgtttga ccacattgcc catgagttcc atgctcagag gc 42 1338 DNA Oligonucleotide 13 gatcctgggg ctggagtggg atggatgatg tcagctgg 3814 40 DNA Oligonucleotide 14 gcgggcagag gatcctgggg ctgtctggcc tcacagcact40 15 236 DNA Human SOM175 15 atgaggggcc aggtacgtga ggtctcccacaggcccctgg aaagaatact tacatctgct 60 cccatggtgt atgcaggtcc gagatgctgaatacagatcc tcatgcaggt gtcaggcaac 120 ttttcaagac ctaaagacag gtgagtctttctcctccgta ggctgcctcc agccccaggc 180 cccccactcc agccccagac ccagacacctgtagccctgc tcaggtgccg aggtga 236 16 1242 DNA mVRF CDS (166)..(789) 16gcacgagctc aggccgtcgc tgcggcgctg cgttgcgctg cctgcgccca gggctcggga 60gggggccgcg gaggagccgc cccctgcgcc ccgccccggg tccccgggtc cgcgccatgg 120ggcggctctg gctgaccccc ccccacaccg ccgggctagg gcccg atg agc ccc ctg 177Met Ser Pro Leu 1 ctg cgt cgc ctg ctg ctt gtt gca ctg ctg cag ctg gctcgc acc cag 225 Leu Arg Arg Leu Leu Leu Val Ala Leu Leu Gln Leu Ala ArgThr Gln 5 10 15 20 gcc cct gtg tcc cag ttt gat ggc ccc agt cac cag aagaaa gtg gtg 273 Ala Pro Val Ser Gln Phe Asp Gly Pro Ser His Gln Lys LysVal Val 25 30 35 cca tgg ata gac gtt tat gca cgt gcc aca tgc cag ccc agggag gtg 321 Pro Trp Ile Asp Val Tyr Ala Arg Ala Thr Cys Gln Pro Arg GluVal 40 45 50 gtg gtg cct ctg agc atg gaa ctc atg ggc aat gtg gtc aaa caacta 369 Val Val Pro Leu Ser Met Glu Leu Met Gly Asn Val Val Lys Gln Leu55 60 65 gtg ccc agc tgt gtg act gtg cag cgc tgt ggt ggc tgc tgc cct gac417 Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly Cys Cys Pro Asp 7075 80 gat ggc ctg gaa tgt gtg ccc act ggg caa cac caa gtc cga atg cag465 Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln Val Arg Met Gln 8590 95 100 atc ctc atg atc cag tac ccg agc agt cag ctg ggg gag atg tccctg 513 Ile Leu Met Ile Gln Tyr Pro Ser Ser Gln Leu Gly Glu Met Ser Leu105 110 115 gga gaa cac agc caa tgt gaa tgc aga cct aaa aaa aag gag agtgct 561 Gly Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys Lys Glu Ser Ala120 125 130 gtg agg cca gac agg gtt gcc ata ccc cac cac cgt ccc cag ccccgc 609 Val Arg Pro Asp Arg Val Ala Ile Pro His His Arg Pro Gln Pro Arg135 140 145 tct gtt ccg ggc tgg gac tct acc ccg gga gca ccc tcc cca gctgac 657 Ser Val Pro Gly Trp Asp Ser Thr Pro Gly Ala Pro Ser Pro Ala Asp150 155 160 atc atc cat ccc act cca gcc cca gga tcc tct gcc cgc ctt gcaccc 705 Ile Ile His Pro Thr Pro Ala Pro Gly Ser Ser Ala Arg Leu Ala Pro165 170 175 180 agc gcc gcc aac gcc ctg acc ccc gga cct gcc gtt gcc gctgta gac 753 Ser Ala Ala Asn Ala Leu Thr Pro Gly Pro Ala Val Ala Ala ValAsp 185 190 195 gcc gcc gct tcc tcc att gcc aag ggc ggg gct tagagctcaaccc 799 Ala Ala Ala Ser Ser Ile Ala Lys Gly Gly Ala 200 205agacacctgt aggtgccgga agccgcgaaa gtgacaagct gctttccaga ctccacgggc 859ccggctgctt ttatggccct gcttcacagg gagaagagtg gagcacaggc gtaacctcct 919cagtctggga ggtcactgcc ccaggacctg gaccttttag agagctctct cgccatcttt 979tatctcccag agctgccatc taacaattgt caaggaacct catgtctcac ctcaggggcc 1039agggtactct ctcacttaac caccctggtc aagtgagcat cttctggctg gctgtctccc 1099ctcactatga aaaccccaaa cttctaccaa taacgggatt tgggttctgt tatgataact 1159gtgacacaca cacacactca cactctgata aaagagatgg agacactaaa aaaaaaaaaa 1219aaaaaaaaaa aaaaaaaaaa aaa 1242 17 207 PRT mVRF 17 Met Ser Pro Leu LeuArg Arg Leu Leu Leu Val Ala Leu Leu Gln Leu 1 5 10 15 Ala Arg Thr GlnAla Pro Val Ser Gln Phe Asp Gly Pro Ser His Gln 20 25 30 Lys Lys Val ValPro Trp Ile Asp Val Tyr Ala Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu ValVal Val Pro Leu Ser Met Glu Leu Met Gly Asn Val 50 55 60 Val Lys Gln LeuVal Pro Ser Cys Val Thr Val Gln Arg Cys Gly Gly 65 70 75 80 Cys Cys ProAsp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg MetGln Ile Leu Met Ile Gln Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu MetSer Leu Gly Glu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 LysGlu Ser Ala Val Arg Pro Asp Arg Val Ala Ile Pro His His Arg 130 135 140Pro Gln Pro Arg Ser Val Pro Gly Trp Asp Ser Thr Pro Gly Ala Pro 145 150155 160 Ser Pro Ala Asp Ile Ile His Pro Thr Pro Ala Pro Gly Ser Ser Ala165 170 175 Arg Leu Ala Pro Ser Ala Ala Asn Ala Leu Thr Pro Gly Pro AlaVal 180 185 190 Ala Ala Val Asp Ala Ala Ala Ser Ser Ile Ala Lys Gly GlyAla 195 200 205 18 188 PRT mVRF167 18 Met Ser Pro Leu Leu Arg Arg LeuLeu Leu Val Ala Leu Leu Gln Leu 1 5 10 15 Ala Arg Thr Gln Ala Pro ValSer Gln Phe Asp Gly Pro Ser His Gln 20 25 30 Lys Lys Val Val Pro Trp IleAsp Val Tyr Ala Arg Ala Thr Cys Gln 35 40 45 Pro Arg Glu Val Val Val ProLeu Ser Met Glu Leu Met Gly Asn Val 50 55 60 Val Lys Gln Leu Val Pro SerCys Val Thr Val Gln Arg Cys Gly Gly 65 70 75 80 Cys Cys Pro Asp Asp GlyLeu Glu Cys Val Pro Thr Gly Gln His Gln 85 90 95 Val Arg Met Gln Ile LeuMet Ile Gln Tyr Pro Ser Ser Gln Leu Gly 100 105 110 Glu Met Ser Leu GlyGlu His Ser Gln Cys Glu Cys Arg Pro Lys Lys 115 120 125 Lys Glu Ser AlaVal Arg Pro Asp Ser Pro Arg Ile Leu Cys Pro Pro 130 135 140 Cys Thr GlnArg Arg Gln Arg Pro Asp Pro Arg Thr Cys Arg Cys Arg 145 150 155 160 CysArg Arg Arg Arg Phe Leu His Cys Gln Gly Arg Gly Leu Glu Leu 165 170 175Asn Pro Asp Thr Cys Arg Cys Arg Lys Pro Arg Lys 180 185 19 188 PRThVRF167 19 Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu GlnLeu 1 5 10 15 Ala Pro Ala Gln Ala Pro Val Ser Gln Pro Asp Ala Pro GlyHis Gln 20 25 30 Arg Lys Val Val Ser Trp Ile Asp Val Tyr Thr Arg Ala ThrCys Gln 35 40 45 Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met GlyThr Val 50 55 60 Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg CysGly Gly 65 70 75 80 Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr GlyGln His Gln 85 90 95 Val Arg Met Gln Ile Leu Met Ile Arg Tyr Pro Ser SerGln Leu Gly 100 105 110 Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu CysArg Pro Lys Lys 115 120 125 Lys Asp Ser Ala Val Lys Pro Asp Ser Pro ArgPro Leu Cys Pro Arg 130 135 140 Cys Thr Gln His His Gln Arg Pro Asp ProArg Thr Cys Arg Cys Arg 145 150 155 160 Cys Arg Arg Arg Ser Phe Leu ArgCys Gln Gly Arg Gly Leu Glu Leu 165 170 175 Asn Pro Asp Thr Cys Arg CysArg Lys Leu Arg Arg 180 185 20 71 PRT mVRF186 20 Arg Val Ala Ile Pro HisHis Arg Pro Gln Pro Arg Ser Val Pro Gly 1 5 10 15 Trp Asp Ser Thr ProGly Ala Pro Ser Pro Ala Asp Ile Ile His Pro 20 25 30 Thr Pro Ala Pro GlySer Ser Ala Arg Leu Ala Pro Ser Ala Ala Asn 35 40 45 Ala Leu Thr Pro GlyPro Ala Val Ala Ala Val Asp Ala Ala Ala Ser 50 55 60 Ser Ile Ala Lys GlyGly Ala 65 70 21 71 PRT hVRF186 21 Arg Ala Ala Thr Pro His His Arg ProGln Pro Arg Ser Val Pro Gly 1 5 10 15 Trp Asp Ser Ala Pro Gly Ala ProSer Pro Ala Asp Ile Thr His Pro 20 25 30 Thr Pro Ala Pro Gly Pro Ser AlaHis Ala Ala Pro Ser Thr Thr Ser 35 40 45 Ala Leu Thr Pro Gly Pro Ala AlaAla Ala Ala Asp Ala Ala Ala Ser 50 55 60 Ser Val Ala Lys Gly Gly Ala 6570 22 214 PRT mVEGF188 22 Met Asn Phe Leu Leu Ser Trp Val His Trp ThrLeu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala Lys Trp Ser Gln AlaAla Pro Thr Thr Glu Gly 20 25 30 Glu Gln Lys Ser His Glu Val Ile Lys PheMet Asp Val Tyr Gln Arg 35 40 45 Ser Tyr Cys Arg Pro Ile Glu Thr Leu ValAsp Ile Phe Gln Glu Tyr 50 55 60 Pro Asp Glu Ile Glu Tyr Ile Phe Lys ProSer Cys Val Pro Leu Met 65 70 75 80 Arg Cys Ala Gly Cys Cys Asn Asp GluAla Leu Glu Cys Val Pro Thr 85 90 95 Ser Glu Ser Asn Ile Thr Met Gln IleMet Arg Ile Lys Pro His Gln 100 105 110 Ser Gln His Ile Gly Glu Met SerPhe Leu Gln His Ser Arg Cys Glu 115 120 125 Cys Arg Pro Lys Lys Asp ArgThr Lys Pro Glu Lys Lys Ser Val Arg 130 135 140 Gly Lys Gly Lys Gly GlnLys Arg Lys Arg Lys Lys Ser Arg Phe Lys 145 150 155 160 Ser Trp Ser ValHis Cys Glu Pro Cys Ser Glu Arg Arg Lys His Leu 165 170 175 Phe Val GlnAsp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp 180 185 190 Ser ArgCys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg 195 200 205 CysAsp Lys Pro Arg Arg 210

1. A biologically isolated proteinaceous molecule having the followingcharacteristics: (i) comprises an amino acid sequence having at leastabout 15% similarity but at least about 5% dissimilarity to the sequenceset forth in SEQ ID NO:2; (ii) exhibits at least one property in commonwith vascular endothelial growth factor (VEGF).
 2. A proteinaceousmolecule according to claim 1 wherein the molecule exhibits at least oneof the following properties: (i) an ability to induce vascularendothelial cells; (ii) an ability to interact with flt-1/flk-1 familyof receptors; and/or (iii) an ability to induce cell migration, cellsurvival and/or an increase in intracellular lavels of alkalinephosphatase.
 3. A proteinaceous molecule according to claim 1 or 2wherein said molecule has the capacity to induce astroglialproliferation.
 4. A proteinaceous molecule according to claim 1 whereinsaid molecule is of human origin.
 5. A proteinaceous molecule accordingto claim 1 wherein said molecule is of non-human origin.
 6. Aproteinaceous molecule according to claim 5 wherein said molecule is oflivestock animal, companion animal, laboratory test animal, avian, fishor reptilian origin.
 7. A proteinaceous molecule according to claim 5wherein said molecule is encoded by a gene located at chromosome 11q13.8. A proteinaceous molecule according to claim 1 wherein the percentagesimilarity to SEQ ID NO:2 is at least about 30%.
 9. A proteinaceousmolecule according to claim 1 wherein the percentage similarity to SEQID NO:2 is at least about 40%.
 10. A proteinaceous molecule according toclaim 1 wherein the percentage similarity to SEQ ID NO:2 is at leastabout 60-70%.
 11. A proteinaceous molecule according to claim 1comprising a sequence of amino acids as set forth in SEQ ID NO:4 or apart, fragment, derivative or analogue thereof.
 12. A proteinaceousmolecule according to claim 1 comprising an amino acid sequencesubstantially set forth in SEQ ID NO:6 or a part, fragment, derivativeor analogue thereof.
 13. A proteinaceous molecule according to claim 1comprising an amino acid sequence substantially set forth in SEQ ID NO:8or a part, fragment, derivative or analogue thereof.
 14. A proteinaceousmolecule according to claim 1 comprising an amino acid sequencesubstantially set forth in SEQ ID NO:10 or a part, fragment, derivativeor analogue thereof.
 15. A recombinant molecule having the followingcharacteristics: (i) an amino acid sequence substantially as set forthin SEQ ID NO:4 or having at least about 15% similarity to but at leastabout 5% dissimilarity to the amino acid sequence set forth in SEQ IDNO:2; (ii) exhibits at least one biological property in common withVEGF.
 16. A recombinant molecule having the following characteristics:(i) an amino acid sequence substantially as set forth in SEQ ID NO:6 orhaving at least about 15% similarity to but at least about 5%dissimilarity to the amino acid sequence set forth in SEQ ID NO:2; (ii)exhibits at least one biological property in common with VEGF.
 17. Arecombinant molecule having the following characteristics: (i) an aminoacid sequence substantially as set forth in SEQ ID NO:8 or having atleast about 15% similarity to but at least about 5% dissimilarity to theamino acid sequence set forth in SEQ ID NO:2; (ii) exhibits at least onebiological property in common with VEGF.
 18. A recombinant moleculehaving the following characteristics: (i) an amino acid sequencesubstantially as set forth in SEQ ID NO:10 or having at least about 15%similarity to but at least about 5% dissimilarity to the amino acidsequence set forth in SEQ ID NO:2; (ii) exhibits at least one biologicalproperty in common with VEGF.
 19. A recombinant molecule according toclaim 15 or 16 or 17 or 18 having at least one of the followingproperties: (a) an ability to induce vascular endothelial cells; (b) anability to interact with flt1/flki family of receptors; (c) an abilityto induce cell migration, cell survival and/or increase intracellularlevels of alkaline phosphatase.
 20. A recombinant molecule according toclaim 15 or 16 or 17 or 18 having the capacity to induce astroglialproliferation.
 21. A recombinant molecule according to claim 20 whereinthe molecule comprises an amino acid sequence substantially as set forthin SEQ ID NO:6.
 22. A peptide fragment corresponding to a portion of theamino acid sequence set forth in SEQ ID NO:4 or a derivative or chemicalequivalent thereof.
 23. A peptide fragment according to claim 22 havingthe sequence set forth in SEQ ID NO:6 or a chemical equivalent thereof.24. A peptide fragment according to claim 22 having the sequence setforth in SEQ ID NO:8 or a chemical equivalent thereof.
 25. A peptidefragment according to claim 22 having the sequence set forth in SEQ IDNO:10 or a chemical equivalent thereof.
 26. A nucleic acid moleculecomprising a sequence of nucleotides or complementary to a sequenceencoding a proteinaceous molecule having the following characteristics:(i) comprises an amino acid sequence having at least about 15%similarity but at least about 5% dissimilarity to the sequence set forthin SEQ ID NO:2; (ii) exhibits at least one property in common withvascular endothelial growth factor (VEGF).
 27. A nucleic acid moleculeaccording to claim 26 wherein the proteinaceous molecule exhibits atleast one of the following properties: (i) an ability to induce vascularendothelial cells; (ii) an ability to interact with flt-1/flk-1 familyof receptors; and/or (iii) an ability to induce cell migration, cellsurvival and/or an increase in intracellular lavels of alkalinephosphatase.
 28. A nucleic acid molecule according to claim 27 whereinthe proteinaceous molecule has the capacity to induce astroglialproliferation.
 29. A nucleic acid molecule according to claim 28 whereinsaid molecule encodes an amino acid sequence substantially as set forthin SEQ ID NO:6.
 30. A nucleic acid molecule according to claim 1 whereinsaid molecule is of human origin.
 31. A nucleic acid molecule accordingto claim 1 wherein the percentage similarity to SEQ ID NO:2 is at leastabout 30%.
 32. A nucleic acid molecule according to claim 26 comprisinga nucleotide sequence substantially as set forth in SEQ ID NO:3 orhaving at least 15% similarity thereto or capable of hybridising underlow stringency conditions to a reverse complement of the nucleotidesequence as set forth in SEQ ID NO:3 provided that the nucleotidesequence has at least 15% similarity but at least 30% dissimilarity tothe nucleotide sequence set forth in SEQ ID NO:3.
 33. A nucleic acidmolecule according to claim 26 encoding a murine homologue of human VEGFand comprising a nucleotide sequence substantially as set forth in FIG.9.
 34. A pharmaceutical composition comprising a proteinaceous moleculeaccording to claim 1 or 2 or 3 or 11 and one or more pharmaceuticallyacceptable carriers and/or diluents.
 35. A method for preparing arecombinant molecule having the following characteristics: (i) comprisesan amino acid sequence having at least about 15% similarity but at leastabout 5% dissimilarity to the sequence set forth in SEQ ID NO:2; (ii)exhibits at least one property in common with vascular endothelialgrowth factor (VEGF), said method comprising expressing a nucleic acidmolecule encoding said recombinant molecule by a suitable host grownunder conditions effective to synthesise said recombinant molecule andthen isolating said molecule.
 36. A method according to claim 35 whereinthe nucleic acid molecule comprises a sequence of nucleotides as setforth in SEQ ID NO:3 or having at least 15% similarity thereto or iscapable of hybridising under low stringency conditions to a reversecomplement of the nucleotide sequence as set forth in SEQ ID NO:3provided that the nucleotide sequence has at least 15% similarity but atleast 30% dissimilarity to the nucleotide sequence set forth in SEQ IDNO:3.
 37. A method of inducing astroglial proliferation in a mammal,said method comprising administering to said mammal an effective amountof a recombinant proteinaceous molecule having the characteristics: (i)comprises an amino acid sequence having at least about 15% similaritybut at least about 5% dissimilarity to the sequence set forth in SEQ IDNO:2; (ii) exhibits at least one property in common with vascularendothelial growth factor (VEGF), said administration being for a timeand under conditions sufficient to induce astroglial proliferation. 38.A method according to claim 37 wherein the recombinant proteinaceousmolecule comprises an amino acid sequence substantially as set forth inSEQ ID NO:3 or is a derivative thereof.
 39. A method according to claim37 wherein the recombinant proteinaceous molecule comprises an aminoacid sequence substantially as set forth in SEQ ID NO:6 or is aderivative thereof.
 40. A method of promoting neuronal survival and/orproliferation in a manmnal, said method comprising administering to saidmammal an effective amount of a recombinant proteinaceous moleculehaving the characteristics: (i) comprises an amino acid sequence havingat least about 15% similarity but at least about 5% dissimilarity to thesequence set forth in SEQ ID NO:2; (ii) exhibits at least one propertyin common with vascular endothelial growth factor (VEGF), saidadministration being for a time and under conditions sufficient toinduce astroglial proliferation.
 41. A method according to claim 40wherein the recombinant proteinaceous molecule comprises an amino acidsequence substantially as set forth in SEQ ID NO:3 or is a derivativethereof.
 41. A method according to claim 40 wherein the recombinantproteinaceous molecule comprises an amino acid sequence substantially asset forth in SEQ ID NO:6 or is a derivative thereof.